Molecular Plant Research Article
Genetic and Epigenetic Diversities Shed Light on Domestication of Cultivated Ginseng (Panax ginseng) Ming-Rui Li1,2,3, Feng-Xue Shi1,2,3, Yu-Xin Zhou1,2,3, Ya-Ling Li1, Xin-Feng Wang1,2, Cui Zhang1,2, Xu-Tong Wang1, Bao Liu1,4, Hong-Xing Xiao2,* and Lin-Feng Li1,* 1
Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, #5268 Renmin Street, Changchun 130024, China
2
Institute of Grassland Science, Northeast Normal University, Key Laboratory of Vegetation Ecology, Ministry of Education, Changchun 130024, China
3These
authors contributed equally to this article.
4Co-senior
author
*Correspondence: Lin-Feng Li (
[email protected]), Hong-Xing Xiao (
[email protected]) http://dx.doi.org/10.1016/j.molp.2015.07.011
ABSTRACT Chinese ginseng (Panax ginseng) is a medically important herb within Panax and has crucial cultural values in East Asia. As the symbol of traditional Chinese medicine, Chinese ginseng has been used as a herbal remedy to restore stamina and capacity in East Asia for thousands of years. To address the evolutionary origin and domestication history of cultivated ginseng, we employed multiple molecular approaches to investigate the genetic structures of cultivated and wild ginseng across their distribution ranges in northeastern Asia. Phylogenetic and population genetic analyses revealed that the four cultivated ginseng landraces, COMMON, BIANTIAO, SHIZHU, and GAOLI (also known as Korean ginseng), were not domesticated independently and Fusong Town is likely one of the primary domestication centers. In addition, our results from population genetic and epigenetic analyses demonstrated that cultivated ginseng maintained high levels of genetic and epigenetic diversity, but showed distinct cytosine methylation patterns compared with wild ginseng. The patterns of genetic and epigenetic variation revealed by this study have shed light on the domestication history of cultivated ginseng, which may serve as a framework for future genetic improvements. Keywords: Panax ginseng, domestication, traditional Chinese medicine, cytosine methylation, genetic and epigenetic diversity Li M.-R., Shi F.-X., Zhou Y.-X., Li Y.-L., Wang X.-F., Zhang C., Wang X.-T., Liu B., Xiao H.-X., and Li L.-F. (2015). Genetic and Epigenetic Diversities Shed Light on Domestication of Cultivated Ginseng (Panax ginseng). Mol. Plant. 8, 1612–1622.
INTRODUCTION Domestication has had profound effects on the course of human history and shifted our societies from nomadic hunter-gatherers to a settled agrarian way of life (Diamond, 2002; Doebley et al., 2006; Gross and Olsen, 2010). Modern humans are still reliant on the plants and animals that were domesticated in diverse places throughout the world (Ross-Ibarra et al., 2007; Olsen and Wendel, 2013). For example, cotton is one of the world’s most important fiber crops and has been used for clothing, fine paper, and other purposes for thousands of years (Mansoor and Paterson, 2012). Previous studies based on multiple molecular approaches revealed the independent domestication of four Gossypium species, of which two allotetraploid (DDAA) species, G. barbadense and G. hirsutum, were domesticated
in America and the remaining two diploid (AA) species, G. arboreum and G. herbaceum, are mainly cultivated in Asia and Africa (Wendel and Cronn, 2003; Wendel and Grover, 2015). To date, more than 90% of the world’s cotton production is supplied by modern cultivars of G. hirsutum (Paterson, 2009; Wendel et al., 2009). East Asia is one of the independent centers of domestication and witnessed the domestication of a remarkable set of plants (Doebley et al., 2006; Larson et al., 2014). The domestication of crop plants (e.g., rice) provides staple food for half of the world’s
Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.
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although different landraces of cultivated ginseng show diverse morphological traits, most of them possess an elongated primary root with numerous lateral roots and rootlets (Figure 1D–1G). In addition, wild ginseng can survive in the natural habitat for hundreds of years, whereas the growth cycle of cultivated ginseng is usually no more than 7 years.
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Figure 1. Cultivated Ginseng in the Field and the Root Morphologies of Wild and Cultivated Ginseng. (A) We can judge the age of cultivated ginseng based on the number of compound leaves. The sample shown in (A) is a 6-year-old cultivated ginseng because it has five compound leaves. (B and C) Wild ginseng collected from southern population KD and northern population EL, respectively. (D) Landrace COMMON ginseng from population FS. (E) Landrace BIANTIAO ginseng from population BT. (F and G) Landrace SHIZHU ginseng from population SZ.
population (Sang and Ge, 2007) and has resulted in the formation of a powerful and expansive agricultural economy in East Asia. Chinese ginseng (Figure 1A) is the symbol of traditional Chinese medicine (TCM) and plays an important role in Chinese medicine culture. Etymologically, the pronunciation of ‘‘gin’’ stands for the Chinese character for ‘‘man’’ and ‘‘seng’’ is the equivalent of ‘‘essence’’ (Hu, 1977). As the symbolic herb of TCM, Chinese ginseng has been used in East Asia for thousands of years as a herbal remedy to restore and enhance stamina and capacity to cope with fatigue and physical stress (Goldstein, 1975; Liu and Xiao, 1992; Gillis, 1997; Coon and Ernst, 2002). Chinese ginseng is a deciduous perennial herb within the genus Panax and was domesticated from the wild progenitor P. ginseng C.A. Meyer (Wen and Zimmer, 1996). Wild ginseng was widely distributed in northeastern Asia as late as the beginning of the 20th century (Zhuravlev et al., 2010). Nowadays, however, only a few ginseng plants exist in natural environments due to the over exploitation of wild resources and the destruction of natural habitats (Reunova et al., 2010; Zhuravlev et al., 2010). In contrast, domesticated ginseng is widely cultivated in northeastern China and the Korean peninsula (Xiao et al., 1987). Unlike other domesticated plants such as rice, maize, and wheat, cultivated ginseng did not undergo drastic morphological modifications and no substantial domestication syndromes have been observed. The major phenotypic differences between wild and cultivated ginseng are the growth cycle and root morphologies, mainly due to human selection acting on the agricultural traits (e.g., root morphologies) and different conditions between natural and artificial growing environments. For example, wild ginseng usually has a short and stout primary root with a slender rhizome and thick branches (Figure 1B and 1C). In contrast,
The genetic diversity and population structure of cultivated ginseng have been reported in previous studies based on random amplified polymorphic DNA (RAPD) (Ma et al., 2000), amplified restriction fragment polymorphism (AFLP) (Ma et al., 2000), and inter-simple sequence repeat (ISSR) (Li et al., 2011). These studies demonstrated that cultivated ginseng harbored a high level of genetic diversity. Similarly, the genetic variability of Russian wild ginseng has also been investigated using allozyme (Koren et al., 2003), RAPD (Zhuravlev et al., 1996), ISSR (Reunova et al., 2010), and AFLP (Zhuravlev et al., 2010). In addition, the epigenetic structure of cultivated and wild ginseng has been evaluated based on methylation-sensitive amplification polymorphism (MSAP) (Ngezahayo et al., 2011). However, the domestication history and genome-wide patterns of genetic and epigenetic variation of Chinese ginseng remain unclear so far due to the limitation of the molecular markers used and the scarcity of wild ginseng samples. Here, we addressed the origin and domestication of cultivated ginseng by integrating six microsatellites, 720 methylation insensitive polymorphism (MISP) loci, and four chloroplast genes of wild and cultivated ginseng across their distribution ranges of northeastern Asia. To further infer the domestication process of cultivated ginseng at genome-wide level, we have sequenced the whole genomes of 18 representative wild and cultivated ginseng accessions from Russian Far East, northeastern China, and the northern Korean peninsula. In addition, two accessions of the landrace GAOLI (also known as Korean ginseng) were downloaded from GenBank to represent the cultivated ginseng of the southern Korean peninsula. To evaluate if domestication has shaped the patterns of genetic and epigenetic variation, we investigated the nucleotide diversity and cytosine methylation pattern of cultivated and wild ginseng based on 49 nuclear genes and 720 methylation-sensitive polymorphism (MSP) loci. The aims of this study were to (1) address where and how cultivated ginseng was domesticated; (2) evaluate the influences of domestication on the patterns of genetic and epigenetic variation of cultivated ginseng. We expect our findings to provide novel insights into the history and domestication of Chinese ginseng.
RESULTS Phylogenetic and Population Genetic Analyses Scanning of the chloroplast genome based on 24 universal primer pairs (Supplemental Table 1) revealed that there are no nucleotide variations within the three wild ginseng samples examined. In addition, we also compared the sequences of the 24 chloroplast genes with the published chloroplast genomes of the landrace COMMON (GenBank: KC686331 and KC686332) and GAOLI (GenBank: KC686333) of cultivated ginseng. Our results showed that no polymorphisms were observed between the wild and cultivated ginseng. In contrast, different haplotypes were observed among the congeneric species of Panax. We therefore reconstructed the neighbor-joining (NJ) tree
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Figure 2. Population-Based UPGMA Tree of Cultivated Ginseng and the Wild Relatives Based on the Six Microsatellites. Blue, cultivar accessions; red, wild accessions; black, other Panax species. LN includes the wild accessions from southern populations CY, HR, and KD. The population names of the remaining wild and cultivated ginseng are the same as in Supplemental Table 4. PN, Panax notoginseng; PQ, Panax quinquefolius; PS, Panax stipuleanatus; PZ, Panax zingiberensis.
(Supplemental Figure 1) based on matK-trnK, rps16, psbI-psbK, and psbA-trnH genes. The phylogenetic tree demonstrated that cultivated and wild ginseng formed a monophyletic clade and differed from the other congeneric species. These observations suggested that the wild form of P. ginseng is the putative progenitor of cultivated ginseng. To further reveal the domestication process of cultivated ginseng, we employed six microsatellites and 720 MISP loci to investigate the genetic structures of wild and cultivated ginseng at the population level. The population-based UPGMA (unweighted pairgroup method using arithmetic averages) tree (Figure 2) and STRUCTURE (Figure 3, K = 2–4) based on microsatellites revealed that all cultivated and wild ginseng populations formed a monophyletic clade and showed distinct genetic clusters from the other Panax species. These results confirmed that cultivated ginseng was indeed domesticated from P. ginseng. In addition, the population-based UPGMA tree (Figure 2) showed that all cultivated ginseng populations were clustered together with the wild ginseng population XL (Fusong Town). Similar topology was also observed in the individual-based UPGMA tree based on the MISP dataset (Supplemental Figure 2A online) where the entire cultivar accessions were grouped together as a monophyletic clade. These findings together suggested the possibility of a single origin of cultivated ginseng and Fusong Town is likely one of the potential domestication centers of cultivated ginseng. It should be noted that although the four landraces GAOLI, BIANTIAO, SHIZHU, and COMMON showed obvious differences in root morphologies and geographic distributions, the phylogenetic trees based on microsatellites (Figure 2) and MISP (Supplemental Figure 2A online) demonstrated that accessions of the four cultivated ginseng landraces were mixed together. Similarly, the STRUCTURE analyses revealed only slight genetic differences among the four landraces (Figure 3, K = 4
and Supplemental Figure 3A, K = 4 online). To gain a genomewide view of the origin and domestication of the four ginseng landraces, we have sequenced the whole genomes of 18 representative wild and cultivated ginseng accessions and downloaded the sequences of two southern Korean peninsula cultivated ginseng accessions from GenBank. A total of 40 387 high-quality single nucleotide polymorphisms (SNPs) were generated from this study all of which were subjected to the phylogenetic and population genetic analyses. As expected, the NJ tree demonstrated that the 11 accessions of the four landraces were mixed together (Figure 4A). We noted that the two southern Korean peninsula accessions showed obviously longer branch length than those of the remaining nine cultivated accessions, suggesting that the two GAOLI accessions possessed more nucleotide substitutions. Likewise, results from the STRUCTURE analysis revealed that although the genetic differences between the Chinese and northern Korean peninsula accessions are not particularly significant, the two southern Korean peninsula accessions exhibited a distinct genetic cluster (Figure 4B). We noted that the 11 cultivated ginseng accessions fall into two distinct clades in the NJ tree (Figure 4A). For instance, three accessions of the landrace COMMOM, BIANTIAO, and SHIZHU were clustered together with the wild ginseng accessions from populations XL and SJ (Fusong Town) (Figure 4A). Likewise, the remaining eight cultivated ginseng accessions were grouped together with the wild ginseng accession from population SJ. These findings based on genome-wide SNPs supported the hypothesis that Fusong Town is the one of the potential domestication centers, but it also implicated the possibility of multiple origins of cultivated ginseng.
Nucleotide Diversity and Demographic History Nucleotide diversity of cultivated and wild ginseng was evaluated at both population and genome-wide levels, respectively. In brief, the 20 representative accessions have generated a total of 40 387 SNPs, of which 37 201 and 28 123 were identified in cultivated and wild ginseng, respectively. The nucleotide diversity of cultivated ginseng at total sites (qT = 0.0093) was slightly higher than that of wild ginseng (qT = 0.0076). In addition, a total of 49 nuclear genes were analyzed for the eight cultivated and wild ginseng libraries separately. The concatenated sequence of the 49 nuclear genes yielded a total of 32 332 base pairs (bp), of which 3179 variable sites were identified. Comparisons of nucleotide diversity for the combined dataset revealed that the wild and cultivated ginseng showed no significant differences in nucleotide diversity at total sites (t-test, p = 0.890 for qT and p = 0.292 for pT, respectively) (Figure 5A and Supplemental Data 1). In addition, our results also showed that nucleotide diversity of the 49 nuclear genes varied dramatically at nonsynonymous, synonymous, and total sites (t-test, all p values
